Platelet activation in the presence of neutral protamine Hagedorn insulin: a new feature of antibodies against protamine/heparin complexes.

Essentials Protamine (PRT) is used to stabilize insulin in neutral protamine Hagedorn (NPH) insulin. The interaction between NPH-insulin, anti-PRT/heparin antibodies and platelets was investigated. Anti-PRT/heparin antibodies activate platelets in presence of NPH-insulin dependent on heparin. Cross-reactivity seems to have no major effect on the clinical outcome of medical patients. SUMMARY: Background Protamine (PRT) is used to stabilize insulin in neutral protamine Hagedorn (NPH) insulin, a commonly used therapeutic agent for diabetes mellitus. Immunization against PRT/heparin complexes is common in diabetic patients. Objectives To investigate the impact of NPH-insulin on the interaction between anti-PRT/heparin antibodies and platelets. Methods The interaction between NPH-insulin and anti-PRT/heparin antibodies was tested using in-house enzyme immunoassays. The ability of anti-PRT/heparin antibodies to activate platelets in the presence of NPH-insulin (and heparin) was investigated using flow cytometry. Results Twenty-one out of 80 sera containing anti-PRT/heparin IgG showed binding to NPH-insulin. Anti-PRT/heparin IgG from immunized patients bound to platelets in the presence of NPH-insulin, but not in the presence of native insulin. Anti-PRT/heparin antibodies induced P-selectin expression in the presence of NPH-insulin in a heparin-dependent way (median mean fluorescence intensity in the presence of NPH-insulin: 55, 95% confidence interval [CI] 18.7-100.5 vs. NPH-insulin and heparin: 204, 95% CI 106.5-372.8). The clinical relevance of platelet-activating anti-PRT/heparin antibodies was assessed by investigating a multicenter study cohort of 332 acutely ill medical patients who received heparin. None of the 21 patients with anti-PRT/heparin IgG developed thrombocytopenia or thromboembolic complications. Conclusions Anti-PRT/heparin antibodies activate platelets in the presence of NPH-insulin in a heparin-dependent way. However, results from our preliminary study indicate no major impact of these antibodies on the clinical outcome in medical patients receiving heparin, particularly on thromboembolic complications.

Protamine (heparin)-induced thrombocytopenia: a review of the serological and clinical features associated with anti-protamine/heparin antibodies.

Protamine is widely used in medicine as a rapidly-acting antidote to heparin, particularly for reversing heparin anticoagulation after cardiac surgery. Protamine is also used as a stabilizing additive to certain preparations of insulin. Recent reports demonstrate that protamine and heparin form multimolecular complexes that result in high rates of immunization in post-cardiac surgery patients, particularly of immunoglobulin G (IgG) class antibodies; a subset of these anti-protamine/heparin IgG antibodies activates platelets through their FcγIIA (IgG) receptors. Although the clinical consequences of anti-protamine/heparin antibodies that are newly generated after cardiac surgery are unknown, there is evidence that platelet-activating anti-protamine/heparin antibodies already present at the time of cardiac surgery might occasionally explain more severe thrombocytopenia with delayed platelet count recovery, as well as thromboembolic complications, in the post-cardiac surgery setting. Triggers for such antibodies remain poorly-defined, but could include preoperative administration of heparin to diabetic patients receiving protamine-insulin as well as recent previous cardiac surgery. Anti-protamine/heparin antibodies have several features in common with anti-platelet factor 4 (PF4) PF4/heparin antibodies implicated in heparin-induced thrombocytopenia (HIT), including immunization by heparin-containing multimolecular complexes, predominant IgG class, pathological platelet-activating properties, relatively rapid IgG formation without IgM precedence, and antibody transience. Despite these similarities, the risk of anti-protamine/heparin antibody-mediated complications seems to affect the early post-cardiac surgery period, whereas HIT usually occurs at least 5 days following cardiac surgery. Clinicians need to become aware of this recently recognized immunohematological disorder, and research is needed to identify triggers of immunization, improve detection of pathological antibodies and identify patients at risk of this complication.